Identifying and quantitating unknown compounds is difficult for both scientists and bioanalytical instruments. Hence, identifying the most sensitive techniques is crucial in early drug development and biotech drug development studies. LC-MS is the most common bioanalytical technique used to detect analytes in complex mixtures. Researchers predominantly use LC-MS/MS analysis to identify and quantify analytes with high sensitivity.
In lc ms ms analysis, the liquid chromatography component separates the target compound while the mass spectrometry unit breaks it down for identification and quantitation. While bioanalytical scientists are familiar with LC-MS/MS analysis, LC-MS method validation and analysis requires an in-depth understanding of the operating conditions and analytes of interest. This guide provides ten tips for a fruitful LC-MS/MS analysis and method validation.
Form a robust validation plan
Before beginning with LC-MS/MS validation, technicians should compile a clear plan consisting of planned experiments, the reason for assay validation, and expected study outcomes. Besides, this plan must align with the guidelines proposed by regulatory agencies.
Understand individual validation cases
Generally, regulatory bodies recommend separate evaluations of performance parameters. However, full method validation for bioanalytical assays is usually very general. Hence, understanding each case is necessary, as, for example, clients may have specific assay requirements.
Focus on method selectivity and analyte stability
Analyte stability and method selectivity are the most crucial parameters, as the remaining parameters largely depend on these two. Hence, technicians should begin validation by assessing method selectivity and analyte stability. Generally, these steps should be followed by linearity, with robustness studies as the last validation steps.
Understand the extent of LC-MS method validation
Depending on study conditions, bioanalytical scientists can determine the extent of LC-MS method validation. Partial validation is doable when small changes are made to a standard protocol. However, adequate evidence is necessary to justify partial method validation.
Sample pretreatment is a vital step when analyzing target compounds at low concentrations. Hence, researchers should focus on removing non-target components to reduce matrix interferences and enhance the S/N ratio for target analytes.
Mobile-phase composition is crucial for ionization and retention of target analytes. Technicians must use highly pure additives and solvents to increase MS background and prevent unwanted adduct formation. Likewise, researchers must use only ultrapure water for mobile-phase preparation.
The interaction between the column and mobile phase controls compound retention. Selecting an ideal column depends on the chemical and physical properties of the target analyte. Hence, technicians must focus on column specification to obtain accurate LC separation.
The calibration curve verifies the true nature of the optimized conditions. Hence, LC-MS analysis should resonate with a nice resolved peak to ensure that the instrument is ready for analyzing the study compound.
Dilution of the chemical standard
A chemical standard free from other compounds ensures optimization free from interferences. Hence, researchers should choose a pure chemical standard for optimization. Depending on the instrument, the standard must be diluted in an appropriate solvent that dissolves the compound without damaging the instrument.
Adequate compound optimization involves the generation of specific energies to direct the analyte for detection and fragmentation. The generated signal from the spectrum largely depends on MS/MS optimization. MS/MS optimization ensures that the generated signal comes from the target analyte and not something else.